GATA6 mutations cause human cardiac outflow tract defects by disrupting semaphorin-plexin signaling Kazuki Kodoa,b, Tsutomu Nishizawab, Michiko Furutanib,c, Shoichi Araib, Eiji Yamamurac, Kunitaka Jood, Takao Takahashia, Rumiko Matsuokab,c,e,1, and Hiroyuki Yamagishia,b,1 aDepartment of Pediatrics, Division of Pediatric Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan; bInternational Research and Educational Institute for Integrated Medical Sciences, eInstitute of Advanced Biomedical Engineering and Science, Graduate School of Medicine, and cDivision of Pediatric Cardiology, Tokyo Women’s Medical University, 8-1, Kawadacho, Shinjyuku-ku, Tokyo 162-8666, Japan; and dDepartment of Pediatrics, Kyushu Koseinenkin Hospital, 1-8-1 Kishinoura Yahatanishi-ku, Kitakyushu 806-8501, Japan Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 1, 2009 (received for review April 30, 2009) Congenital heart diseases (CHD) occur in nearly 1% of all live births fects, typically PTA (9–12). More recent studies have shown that and are the major cause of infant mortality and morbidity. Al- reciprocal signaling between the CNC and cells derived from the though an improved understanding of the genetic causes of CHD pharyngeal mesoderm (or second heart field), which give rise to would provide insight into the underlying pathobiology, the ge- OFT/subpulmonary myocardium, may also be required for OFT netic etiology of most CHD remains unknown. Here we show that development (13–15). mutations in the gene encoding the transcription factor GATA6 Genes encoding members of the GATA family of zinc finger cause CHD characteristic of a severe form of cardiac outflow tract transcription factors, specifically GATA4, GATA5, and GATA6, (OFT) defect, namely persistent truncus arteriosus (PTA). Two restrict the developmental potential of multiple distinct cell lineages different GATA6 mutations were identified by systematic genetic and regulate morphogenetic patterning in the embryo, including analysis using DNA from patients with PTA. Genes encoding the formation of the heart (16, 17). Null mutation of Gata4 in mice neurovascular guiding molecule semaphorin 3C (SEMA3C) and its results in embryonic lethality because of defects in cardiogenesis receptor plexin A2 (PLXNA2) appear to be regulated directly by (18, 19). Mutations of GATA4 in humans were identified to cause GATA6, and both GATA6 mutant proteins failed to transactivate CHD characteristic of atrial and/or ventricular septal defects (20, GENETICS these genes. Transgenic analysis further suggests that, in the 21), and heterozygous mutations of Gata4 in mice recapitulate the developing heart, the expression of SEMA3C in the OFT/subpul- human phenotype (22). Systemic ablation of Gata6 in mice also monary myocardium and PLXNA2 in the cardiac neural crest con- results in embryonic lethality (23), whereas conditional inactivation tributing to the OFT is dependent on GATA transcription factors. of Gata6 in the CNC results in perinatal mortality from OFT Together, our data implicate mutations in GATA6 as genetic causes defects, typically PTA (24). These observations suggest that defects of CHD involving OFT development, as a result of the disruption of in GATA6 and its downstream target genes that regulate CNC the direct regulation of semaphorin-plexin signaling. development could be responsible for human CHD involving the OFT, however, no mutations in GATA6 have yet been reported in congenital heart disease ͉ persistent truncus arteriosus ͉ humans. cardiac neural crest To develop a systematic genetic analysis for the etiology of various cardiovascular diseases, we have established a genomic bank of over 3,000 Japanese patients with cardiovascular diseases. ongenital heart diseases (CHD) constitute a major percentage This bank contains 21 genomes from independent Japanese pa- Cof clinically significant birth defects with an estimated preva- tients with non-syndromic PTA and, after screening the 21 ge- lence of 4–10 per 1,000 live infants (1). Cardiac outflow tract (OFT) nomes, we identified two mutations of GATA6 responsible for defects are estimated to account for approximately 30% of CHD (2) PTA. Both mutations were reconfirmed using patients’ original and usually require an intervention during the first year of life. A genomic DNA. We also showed that GATA6 directly regulated variety of OFT defects results from disturbance of the morphoge- genes encoding the neurovascular guiding molecule semaphorin 3C netic patterning of the anterior pole of the heart, which is essential (SEMA3C), as well as its receptor plexin A2 (PLXNA2), involved for the establishment of separate systemic and pulmonary circula- in the development of the CNC. Both GATA6 mutant proteins tions in higher vertebrates. Persistent truncus arteriosus (PTA), failed to transactivate gene expression of SEMA3C and PLXNA2, which is attributed to missing septation of the OFT, is recognized suggesting an underlying genetic and molecular basis for OFT as the most severe phenotype of OFT defect, and is often associated defect. with an unfavorable prognosis because complete surgical repair is not always possible (3). Although an improved understanding of Results possible genetic causes would provide insight into the pathogenesis GATA6 Mutations Were Identified in Patients with PTA. Two GATA6 of CHD and allow for better assessment of disease risk, prenatal mutations were detected and confirmed in two independent pro- diagnosis, and critical information for disease prevention, the bands with PTA (Fig. 1A, Table 1 and Table S1). A frame shift etiology of most CHD, including OFT defects, remains unknown because of the multifactorial nature of the diseases (4–6). Based on animal studies, it appears that abnormal development Author contributions: K.K., T.T., R.M., and H.Y. designed research; K.K., T.N., M.F., S.A., E.Y., of cardiac neural crest (CNC) cells, an ectoderm-derived cell K.J., and R.M. performed research; K.K. and T.N. contributed new reagents/analytic tools; K.K. analyzed data; and K.K. and H.Y. wrote the paper. lineage, contributes significantly to the pathology of OFT defects (7–12). During early embryogenesis, CNC cells arise from the The authors declare no conflict of interest. dorsal neural tube and migrate ventrally as mesenchymal cells to This article is a PNAS Direct Submission. populate the OFT, where they coalesce to form the aorticopulmo- The sequences reported in this paper have been deposited in the GenBank database [accession nos. GATA6 (NM࿝005257), GATA4 (NM࿝002052), SEMA3C (NM࿝006379), PLXNA2 (NM࿝025179), nary septum, which divides the single truncus arteriosus (embryonic NPPA (NM࿝006172), WNT2 (NM࿝003391), Sema3c (NM࿝013657), Plxna2 (NM࿝008882)]. OFT) into the aorta and pulmonary artery, resulting in the estab- 1To whom correspondence may be addressed. E-mail: [email protected] or lishment of separate systemic and pulmonary circulations (7, 8). A [email protected]. number of mouse lines in which the genes implicated in CNC This article contains supporting information online at www.pnas.org/cgi/content/full/ development have been ablated produce offspring with OFT de- 0904744106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904744106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 E486del N466H A human GATA6 B ZfZfZf Zf ZfNLS + - -- N466H* E486del* ++ + A G GG AAAA G A E486del LAMKKGNSNQETKT Ter G G A A T GG CCC A T G human LAMKKEGIQTRKRKP mouse LAMKKEGIQTRKRKP chick LAMKKEGIQTRKRKP Proband xenopus LAMKKEGIQTRKRKP zebrafish LAMKKEGIQTRKRKP AA AA G A G GG T GG C AAT G C nuclear localization signal Control N466H GEPVCH ACGL human GEPVCN ACGL mouse GEPVCN ACGL A G GG chick GEPVCN ACGL AA AA G xenopus GEPVCN ACGL G G A A G T G C AAT G C zebrafish GEPVCN ACGL Father zinc finger AA AA G A G GGG T G C AAT G C C Mother Ao Pa Pa Ao AA G A G GG AA G G A A LV RV Sibling Fig. 1. Identification of GATA6 mutations in patients with persistent truncus arteriosus (PTA). (A) The structure of the human GATA6 gene, the position of the two mutations (asterisks), and conservation of alignment between species are shown. Zf, zinc finger; NLS, nuclear localization signal. Changes in amino acids are highlighted in red. The E486del mutation causes two nucleotide deletions, resulting in nine amino acid changes followed by P489 to termination (Ter) codon. (B) Pedigree indicating cardiac phenotype and the presence (ϩ) or absence (-) of the GATA6 mutation in the family of proband A (E486del; arrow) and proband B (N466H; arrow). The probands are indicated by arrows. (circle), female; (box), male; (solid fill), with CHD. In addition, a sequence chromatogram of one frame shift mutation (E486del, proband A) and a point mutation (N466H, proband B) are shown. (C) A catheter angiogram of proband A reveals a single common outflow tract with no septation between the aorta (Ao) and pulmonary artery (Pa), diagnosed as PTA. A schematic diagram of PTA is shown on the right. RV, right ventricle; LV, left ventricle. mutation (E486del) was detected in proband A. The frameshift a single nucleotide polymorphism (SNP) of GATA6. None of the caused changes in the amino acid sequence, converting P489 to a previously reported SNPs on GATA6 were detected in this Japanese stop codon, disrupting the nuclear localization signal (NLS), and population (Table S1). truncating 100 amino acids at the C terminus (Fig. 1A). Another was point mutation which located in the zinc finger domain in GATA6 Mutations Cause Defects in Nuclear Localization and the proband B (N466H in exon 4). Analysis of family members of Transactivation Ability of the GATA6 Protein. To assess the impact of proband A revealed that identical mutations (E486del) on GATA6 the two mutations on the structural and functional properties of were carried by the proband’s father and sister and that both had GATA6, site-directed mutagenesis was performed on human CHD. However, proband A’s mother had neither mutations of GATA6 cDNA cloned in a FLAG-tagged expression vector.